Journal: STAR Protocols
Article Title: Protocol to annotate dendritic cell maturation types in vivo making use of lipid nanoparticle-based approaches
doi: 10.1016/j.xpro.2026.104395
Figure Lengend Snippet: Panel design For efficiency, staining mixes are prepared one day before the readout. These include mixes for experimental samples, single-stained cell controls, and single-stained bead controls ( C and 2D). Mixes are prepared without cells and added on the day of the readout. The optimized panel uses two sequential staining steps. (A) 96-well plate layout for single-stained beads (B). (B) 96-well plate layout for the staining for the single-stained cell controls, SS1: single-stained staining 1, SS2: singe-stained staining 2. (C) Panel overview and information on staining mixes for the experimental samples, single-stained beads and single-stained cell controls for conventional flow cytometry. (D) Panel overview and information on staining mixes for the experimental samples, single-stained beads and single-stained cell controls for FACS. We assign a number to each marker to simplify labeling of the wells for the single-stained controls. The total volume of the experimental staining mixes for experimental samples is determined by the number of samples to be stained. For single-stained controls, we always prepare the staining in a total volume of 200 μL. All staining mixes and control samples are then prepared according to the required antibody dilutions. For the channel corresponding to the LNPs, single-stained controls consist of cells from a mouse injected with LNPs. For single-stained beads, an antibody conjugated to a fluorochrome detected in the same channel need to be used.
Article Snippet: Add 500 μL of the 2× digestion buffer 1. c. Incubate the Eppendorf tube in a 37°C heat block and put the shaker on maximum for 20 min. d. Resuspend tissue every 10 min. e. Filter over a 5 mL polystyrene FACS tube with filtercap (35 μM), smash, wash Eppendorf tube with 2 mL 1× PBS and filter again over the filtercap (35 μM). f. If enrichment for cDCs is necessary, go further to step 11. g. Remove supernatant and resuspend in 200 μL FACS buffer. h. Count cells.
Techniques: Staining, Flow Cytometry, Marker, Labeling, Control, Injection
